Stimulated secretion of granules from cultured RBL-2H3 mast cells is dependent on mobilization of calcium via phospholipase (PL) C and activation of protein kinase (PK) C along with PLD. In recent years we have focussed on PLD because this enzyme is the primary source of phosphatidic acid and diglycerides derived therefrom (both stimulants of PKC) and because its activation is essential for secretion in stimulated mast cells (see previous reports in this series). In intact cells, PLD is activated by antigen and synergistically by stimulants of CaM kinase II (thapsigargin), PKC (phorbol ester) and PKA (cholera toxin). The activation of PLD by antigen (via the IgE receptor, FcepsilonRI) is partially blocked by inhibitors of each of these kinases. The inhibition is augmented by combinations of inhibitors but a component of PLD activation is resistant to these inhibitors to suggest additional cryptic activation mechanism(s). These studies also showed that activation of PLD, along with an increase in cytosolic calcium ions, was a necessary signal for secretion. For example, in all experiments the extent of activation of PLD and secretion were highly correlated but only when when stimulation was accompanied by a calcium signal. In addition, secretion was suppressed by primary alcohols (but not by secondary and tertiary alcohols)that divert production of the PLD product, phosphatidic acid to phosphatidylalcohol, a reaction referred to as transphosphatidylation. We have now identified one cryptic signalling mechanism for activation of PLD2 (one of two isoforms of PLD) which is located exclusively on the plasma membrane of RBL-2H3 cells (see below). Point mutation of unique tyrosine phosphorylation sites in PLD2 indicate three sites of tyrosine phosphorylation on PLD2, two of which are essential for activation of PLD2 by antigen. This activation/phosphorylation is associated with binding of PLD2 to Lyn, a Src kinase known to initiate signaling cascades in antigen-stimulated mast cells. This association, the phosphorylation/activation of PLD2, and secretion are all blocked by the Src kinase inhibitor PP2 at exactly the same concentrations and by expression PLD2 mutated at either of the two critical tyrosine residues.These studies not only reaffirm an essential requirement of PLD for secretion but also provide the first demonstration of the regulation of PLD2 by a tyrosine kinase and the first defined mechanism of activation of this PLD. Prior to these studies, the mechanism of activation of PLD2 was unclear unlike PLD1 which is known to be activated via small G-proteins such as ARF-1 and Rho as well as by serine/threonine kinases. RBL-2H3 cells express both PLD1 and 2 and our studies show that both isoforms perform diferent but complementary roles in secretion. Expression of wild type and catalytically inactive mutants of PLD1 and 2 fused with enhanced green fluorescent protein (EGFP) indicate that PLD1 and PLD2 are located on secretory granules and the plasmam membrane, respectively. Both isoforms are activated by antigen and synergistically by the stimulants of CaM kinase II, PKC, and PKA noted above. After stimulation of cells, PLD1 migrates along with granules to the cell periphery and fuses with the plasma membrane. Expression of the mutated inactive forms of PLD1 and 2 which distribute exactly as their wild type counterparts blocks migration of granules and their fusion with the plasma membrane, respectively. 1-Butanol but not tertiary butanol also blocks migration and fusion PLD1-labelled granules. It is apparent that both PLD1 and 2 respond to secretory stimuli and participate in the secretory process; PLD1 in the translocation of granules and PLD2, possibly in conjunction with PLD1, in the final fusion events.These results imply that the activation of the PLDs must be coordinated to achieve completion of both processes of the exocytotic process, i.e. migration and fusion of granules. Our assumption is that PLD2 because of its association with the plasma membrane and its demonstrated accessibility to FcepsilonRI associated kinases, namely Lyn, is activated first (our kinetic experiments suggest this to be the case) and that PLD2 is activated downstream of other Fcepsilon mediated pathways. The role of serine/threonine kinases in this process will be the subject of future studies. As mentioned above, PLD is activated by PKC but the presumption that PLD may in turn activate PKC through generation of phosphatidic acid and diglycerides generated from phosphatidic acid is a matter of dispute. However, we find that antigen induced translocation and phosphorylation of various isoforms of PKC is blocked by 1-butanol but not by tertiary butanol as is secretion. As a control, direct activation of the PKC isoforms by phorbol ester is not affected by the butanols. It would appear that PLD activation is indeed essential for PKC activation. We suggest that that PLD and PKC, each of which can activate the other, provide an amplifying loop in the secretory pathway and accounts for the widely reported synergistic interactions of pharmacologic stimulants on mast cell secretion.